Oncogenic gene expression and epigenetic remodeling of cis-regulatory elements in ASXL1-mutant chronic myelomonocytic leukemia

Abstract

Myeloid neoplasms are clonal hematopoietic stem cell disorders driven by the sequential acquisition of recurrent genetic lesions. Truncating mutations in the chromatin remodeler ASXL1 (ASXL1^MT) are associated with a high-risk disease phenotype with increased proliferation, epigenetic therapeutic resistance, and poor survival outcomes. We performed a multi-omics interrogation to define gene expression and chromatin remodeling associated with ASXL1^MT in chronic myelomonocytic leukemia (CMML). ASXL1^MT are associated with a loss of repressive histone methylation and increase in permissive histone methylation and acetylation in promoter regions. ASXL1^MT are further associated with de novo accessibility of distal enhancers binding ETS transcription factors, targeting important leukemogenic driver genes. Chromatin remodeling of promoters and enhancers is strongly associated with gene expression and heterogenous among overexpressed genes. These results provide a comprehensive map of the transcriptome and chromatin landscape of ASXL1^MT CMML, forming an important framework for the development of novel therapeutic strategies targeting oncogenic cis interactions.

Introduction

Chronic myeloid neoplasms are malignant clonal hematopoietic stem cell disorders driven by recurrent genetic events, with an inherent risk of transformation to acute myeloid leukemia (AML)^1,2. Within myeloid neoplasms, chronic myelomonocytic leukemia (CMML) represents an attractive disease model since it is characterized by both myelodysplastic and myeloproliferative features, while retaining a relatively simple clonal composition³. CMML shares the typical repertoire of genetic driver lesions with other myeloid neoplasms and is particularly enriched in truncating mutations involving ASXL1 (prevalence ~40%)³. The presence of truncating ASXL1 mutations in CMML is associated with proliferative disease features, resistance to epigenetic therapies, and adverse outcomes^4,5,6. Due to their independent prognostic significance, ASXL1 mutations have been incorporated in all three contemporary molecularly integrated CMML-specific prognostic models^5,7,8. Given the paucity of effective therapies for CMML, delineating the molecular mechanisms of ASXL1-mutant CMML (ASXL1^MT) is of particular interest from a therapeutic standpoint.

The sum of evidence from mechanistic studies suggests that ASXL1 has a complex interactome, that truncating ASXL1 mutations promote leukemogenesis by transcriptional up-regulation of leukemogenic drivers including posterior HOXA genes, and that these mutations recruit several effectors to alter the epigenome through histone modifications, increases in chromatin accessibility, and remodeling of enhancers^{9,10,11,12,13,14}. However, to our knowledge, no studies have been able to extensively query the epigenome in primary patient samples. Given the complexity of human transcriptional regulation in vivo and the multitude of potential epigenetic mechanisms cooperating to regulate transcriptional activity, questions remain about the interplay of regulatory mechanisms in patients with CMML. To elucidate this interplay, we interrogated the genome, transcriptome, and epigenome of patients with ASXL1-wildtype (ASXL1^WT) and ASXL1^MT CMML. In this work, we integrated somatic mutations, transcription, (hydroxy)methylation, histone modifications, and chromatin accessibility to reveal the complexity of the epigenetic landscape, the simultaneous presence of multiple regulatory mechanisms affecting drivers of leukemogenesis, and remodeling of the enhancer landscape as an important driver of intratumoral heterogeneity. These insights into the epigenetic landscape of ASXL1^MT CMML generated from primary patient samples are of considerable interest for the development of novel targeted therapeutic strategies for patients with ASXL1^MT CMML.

Results

To survey the epigenetic landscape of human CMML, we interrogated mutational spectrum, transcription, DNA methylation, histone modifications, and chromatin accessibility in ASXL1^MT (n = 8) and ASXL1^WT (n = 8) CMML (Fig. 1a). The clinical characteristics of the 16 patients with WHO-defined CMML included in this study are shown in Table 1 and Supplementary Data 2. All mutations in ASXL1 resulted in a frameshift and were predicted to lead to a truncation of the protein’s plant homeodomain (Fig. 1b). The spectrum of co-mutations was consistent with previous observations and included spliceosome components, chromatin regulators, modulators of DNA methylation, and cell signaling molecules (Fig. 1c). Abnormal karyotypes were observed in the same number of patients and the burden of co-mutations was similar between the two groups (median number per group 3 versus 3, p = 0.508). This included several modulators of DNA methylation including TET2, DNMT3A, and IDH2 (median number per group 1 versus 1, p = 0.699). As previously reported, G646W (c.1934dup) was the most prevalent ASXL1 mutation and the observed variant allele frequencies of all ASXL1 mutations were consistent with heterozygosity^15,16. The presence of truncating ASXL1 mutations was associated with increased all-cause mortality in the larger patient population seen at our institution (n = 375) from which the 16 patients is this study were sampled (Fig. 1d, Supplementary Fig. 1a, b). For additional information on patient, sample, and cell selection, please refer to Methods and Supplementary Fig. 1c.